Method to prepare compositions comprising yeast treated with electromagnetic energy

ABSTRACT

The present invention relates to pharmaceutical compositions and dietary supplement comprising yeast cells that can produce a healthful benefit in a subject inflicted with uterine cancer. The biological compositions can be used to retard the growth of uterine cancer cells and/or prolonging the time of survival of the subject. The invention also relates to methods for manufacturing the biological compositions.

1. FIELD OF THE INVENTION

The invention relates to oral compositions comprising yeast cells thatcan produce a healthful benefit in a subject inflicted with uterinecancer. The invention also relates to methods for manufacturing the oralcompositions and methods of use thereof.

2. BACKGROUND OF THE INVENTION

2.1 Uterine Cancer

Endometrial cancer, or cancer in the lining (endometrium), is the mostcommon type of uterine cancer. Endometrial cancer is the most commonfemale genital cancer and ranks fourth in prevalence behind breast,lung, and colorectal cancers. In 1994, about 46,000 new cases and 10,500deaths in the U.S. were associated with endometrial cancer. (Boring etal., 1994, CA Cancer J Clin. 44:7–26). The incidence of endometrialcancer in women in the United States is 1% to 2%. It affects mostlypostmenopausal women with a median age of 58 years. However, 25% ofcases occur in premenopausal women. The incidence of endometrial cancervaries widely among different ethnic groups and countries, and arehighest for whites and lowest for blacks and Filipinos.

Increased risk of developing endometrial cancer has been noted in womenwith increased levels of natural estrogen or those subject to long termhormone replacement therapy. Associated conditions include obesity,diabetes, hypertension, and polycystic ovarian disease. Studies haveshown that women taking the drug Tamoxifen to prevent or treat breastcancer have an increased risk of uterine cancer. Other risk factors suchas nulliparity (never having carried a pregnancy), infertility, earlymenarche (onset of menstruation), and late menopause (cessation ofmenstruation) also increases a woman's risk of being diagnosed withendometrial cancer. Women who have a history of endometrial hyperplasia,endometrial polyps or other benign growths of the uterine lining,postmenopausal women who use estrogen-replacement therapy (specificallyif not given in conjunction with periodic progestin), and women withdiabetes also fit into the higher risk category.

The most common symptom of uterine cancer is abnormal vaginal and/oruterine spotting or bleeding, such as between periods or afterintercourse. Often there is also white or clear vaginal discharge,difficult or painful urination, pain or cramping in the pelvic area, anddiscomfort during intercourse. However, there are often no symptoms ofuterine cancer until the disease is advanced. Even then, symptoms suchas loss of appetite, weight loss, fatigue, back or leg pain, leaking ofurine or feces from the vagina, and bone fracture are vague andnonspecific and may be caused by other diseases. In advanced cases,endometrial cancer may present like ovarian cancer with ascites, pelvicor abdominal mass, and bowel symptoms. Screening procedures commonlyused are pelvic exam, Papanicolaou (Pap) smear, transvaginal ultrasound,and biopsy.

Endometrial cancer usually develops in the uterine fundus and grows intothe endometrial cavity, producing an exophytic lesion. It is generallyaccepted that endometrial hyperplasia results from unopposed estrogenstimulation. The most common histologic type of endometrial cancer isendometrioid carcinoma, accounting for 75% to 80% of case. Squamousdifferentiation occurs in one third to one half of cases.Well-differentiated lesions correspond to adenoacanthoma, and poordifferentiation to adenosquamous carcinoma. Other types of endometrialcancer include papillary serous carcinomas (10%) and clear celladenocarcinomas (4%).

The staging of endometrial cancer is based on the revised criteria ofTNM staging by the American Joint Committee for Cancer (AJCC) publishedin 1988. Staging is the process of describing the extent to which cancerhas spread from the site of its origin. It is used to assess a patient'sprognosis and to determine the choice of therapy. The stage of a canceris determined by the size and location in the body of the primary tumor,and whether it has spread to other areas of the body. Staging involvesusing the letters T, N and M to assess tumors by the size of the primarytumor (T); the degree to which regional lymph nodes (N) are involved;and the absence or presence of distant metastases (M)—cancer that hasspread from the original (primary) tumor to distant organs or distantlymph nodes. Each of these categories is further classified with anumber 1 through 4 to give the total stage. Once the T, N and M aredetermined, a “stage” of I, II, III or IV is assigned. Stage I cancersare small, localized and usually curable. Stage II and III cancerstypically are locally advanced and/or have spread to local lymph nodes.Stage IV cancers usually are metastatic (have spread to distant parts ofthe body) and generally are considered inoperable.

Uterine cancer can be treated with surgery, radiation therapy,chemotherapy, surveillance, adjuvant (additional), or a combination ofthese treatments. Treatment of uterine cancer depends on the type ofcancer, the stage, the size and shape of the tumor, the age and generalhealth of the woman, and her desire for future childbearing.

There are many different types of surgery for uterine cancer. Most womenwith uterine cancer have surgery to remove the uterus (hysterectomy).The doctor also removes both fallopian tubes and both ovaries (bilateralsalpingo-oophorectomy). The doctor may also remove the lymph nodes nearthe tumor to see if they contain cancer. Radial hysterectomy isassociated with a 5-year survival of 90.1%. (Morrow CP and Townsend DE,1987, Synopsis of Gynecologic Oncology. 3rd ed. New York, N.Y.: JohnWiley & Sons Inc., p. 107). A cone biopsy or trachelectomy may be usedif the cancerous cells have spread only very slightly beyond the surfacecells of the uterine. In certain situations, the ovaries may also beremoved but, where possible, they are not taken out in young women astheir removal brings on an early menopause. In the most extreme surgery,called a pelvic exenteration, all of the organs of the pelvis, includingthe bladder and rectum, are removed.

Postoperative radiation is always given in fractionation, to a totaldose of 4,500 to 5,000 rads. Even though no survival advantage wasshown, radiation therapy appeared to reduce the incidence of pelvicrecurrences. Radiation is also used to shrink an especially large tumorprior to surgery or to slow the growth of inoperable tumors using eitherexternal beam (similar to an x-ray) or brachytherapy (internal radiationdelivered with implanted radioactive seeds). Fatigue is a possible sideeffect of radiation therapy, but it gradually ceases after treatment iscompleted.

Short-term chemotherapy, such as hydroxyurea and cisplatin, is usedprimarily in cases where the disease has spread outside the uterine andwhere hormonal treatments alone are no longer effective in preventingtumor growth. Potential side effects include nausea and vomiting, lossof hair, low blood cell counts, and fatigue. Many chemotherapeutic drugshave been tried in the past as single agents for the palliation ofuterine cancer, but the results were generally disappointing.Nevertheless, the role of chemotherapy in the management of uterinecancer is continually evolving. Oftentimes, chemotherapy with radiationin adjunct to surgery is used. In general, chemotherapy can achievelong-term survival rates of up to 15% to 20%, even in patients withrecurrent or metastatic disease (Ali et al., 2000, Oncology14(8):1223–30). Unfortunately, the high initial response rates to firstline chemotherapy does not appear to translate into a survival benefit(Kohno and Kitahara, 2001, Gan To Kagaku Ryoho 28(4):448–53). Moreover,there are many undesirable side effects associated with chemotherapysuch as temporary hair loss, mouth sores, anemia (decreased numbers ofred blood cells that may cause fatigue, dizziness, and shortness ofbreath), leukopenia (decreased numbers of white blood cells that maylower resistance to infection), thrombocytopenia (decreased numbers ofplatelets that may lead to easy bleeding or bruising), andgastrointestinal symptoms like nausea, vomiting, and diarrhea. Activechemotherapeutic agents include cyclosporin A, Taxol, Cisplatin,Carboplatin, Adriamycin, Doxil and Topotecan.

The identification of active chemotherapeutic agents against cancerstraditionally involved the use of various animal models of cancer. Themouse has been one of the most informative and productive experimentalsystem for studying carcinogenesis (Sills et al., 2001, Toxicol Letters120:187–198), cancer therapy (Malkinson, 2001, Lung Cancer32(3):265–279; Hoffmnan R M., 1999, Invest New Drugs 17(4):343–359), andcancer chemoprevention (Yun, 1999, Annals NY Acad Sci. 889:157–192).Cancer research started with transplanted tumors in animals whichprovided reproducible and controllable materials for investigation.Pieces of primary animal tumors, cell suspensions made from thesetumors, and immortal cell lines established from these tumor cellspropagate when transplanted to animals of the same species.

To transplant human cancer to an animal and to prevent its destructionby rejection, the immune system of the animal are compromised. Whileoriginally accomplished by irradiation, thymectomy, and application ofsteroids to eliminate acquired immunity, nude mice that are athymiccongenitally have been used as recipients of a variety of human tumors(Rygaard, 1983, in 13^(th) International Cancer Congress Part C, Biologyof Cancer (2), pp37–44, Alan R. Liss, Inc., NY; Fergusson and Smith,1987, Thorax, 42:753–758). While the athymic nude mouse model providesuseful models to study a large number of human tumors in vivo, it doesnot develop spontaneous metastases and are not suitable for all types oftumors. Next, the severe combined immunodeficient (SCID) mice isdeveloped in which the acquired immune system is completely disabled bya genetic mutation. Human lung cancer was first used to demonstrate thesuccessful engraftment of a human cancer in the SCID mouse model (ReddyS., 1987, Cancer Res. 47(9):2456–2460). Subsequently, the SCID mousemodel have been shown to allow disseminated metastatic growths for anumber of human tumors, particularly hematologic disorders and malignantmelanoma (Mueller and Reisfeld, 1991, Cancer Metastasis Rev. 10(3):193–200; Bankert et al., 2001, Trends Immunol. 22:386–393). With therecent advent of transgenic technology, the mouse genome has become theprimary mammalian genetic model for the study of cancer (Resor et al.,2001, Human Molec Genet. 10:669–675).

While surgery, chemotherapeutic agents and radiation are useful in thetreatment of uterine cancer, there is a continued need to find bettertreatment modalities and approaches to manage the disease that are moreeffective and less toxic, especially when clinical oncologists aregiving increased attention to the quality of life of cancer patients.The present invention provides an alternative approach to cancer therapyand management of the disease by using an oral composition comprisingyeasts.

2.2 Yeast-Based Compositions

Yeasts and components thereof have been developed to be used as dietarysupplement or pharmaceuticals. However, none of the prior methods usesyeast cells which have been cultured in an electromagnetic field toproduce a product that has an anti-cancer effect. The following are someexamples of prior uses of yeast cells and components thereof:

U.S. Pat. No. 6,197,295 discloses a selenium-enriched dried yeastproduct which can be used as dietary supplement. The yeast strainSaccharomyces boulardii sequela PY 31 (ATCC 74366) is cultured in thepresence of selenium salts and contains 300 to about 6,000 ppmintracellular selenium. Methods for reducing tumor cell growth byadministration of the selenium yeast product in combination withchemotherapeutic agents is also disclosed.

U.S. Pat. No. 6,143,731 discloses a dietary additive containing wholeβ-glucans derived from yeast, which when administered to animals andhumans, provide a source of fiber in the diet, a fecal bulking agent, asource of short chain fatty acids, reduce cholesterol and LDL, andraises HDL levels.

U.S. Pat. No. 5,504,079 discloses a method of stimulating an immuneresponse in a subject utilizing modified yeast glucans which haveenhanced immunobiologic activity. The modified glucans are prepared fromthe cell wall of Saccharomyces yeasts, and can be administered in avariety of routes including, for example, the oral, intravenous,subcutaneous, topical, and intranasal route.

U.S. Pat. No. 4,348,483 discloses a process for preparing a chromiumyeast product which has a high intracellular chromium content. Theprocess comprises allowing the yeast cells to absorb chromium under acontrolled acidic pH and, thereafter inducing the yeast cells to grow byadding nutrients. The yeast cells are dried and used as a dietarysupplement.

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of theclaims of the present application. All statements as to the date orrepresentations as to the contents of these documents are based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

3. SUMMARY OF THE INVENTION

The present invention relates to biological or oral compositions usefulfor subjects with uterine cancer. In one embodiment, the presentinvention provides biological compositions comprising live yeast cellswhich are capable of producing a healthful benefit in subjects withuterine cancer. In other embodiments, the invention provides methods ofmaking the biological compositions, and methods of using the biologicalcompositions.

In particular, the methods of the invention comprise culturing yeastcells in the presence of a series of electromagnetic fields such thatthe yeast cells becomes metabolically active. The electromagnetic fieldsused are each defined by one of five frequency ranges and a broad rangeof field strength. The starting yeast cells are commercially availableand/or accessible to the public, such as but not limited toSaccharomyces. The methods for making the biological compositions of theinvention further comprise conditioning the activated yeast cells inplant extracts and the gastric juice of animals, while in the presenceof another series of electromagnetic fields.

The methods of manufacturing also comprise expanding the number ofactivated or activated and conditioned yeast cells in large scalecultures in the presence of yet another series of electromagneticfields, performing quality control measures, and packaging.Pharmaceutical compositions of the invention comprises activated andconditioned yeast cells and one or more pharmaceutically acceptableexcipients or carriers. Additional ingredients, such as vitamins and/orflavors may be added to the biological compositions to form the oralcompositions of the invention. Such additional carriers and ingredientscan improve the healthful benefits, pharmacological properties, andorganoleptic characteristics of the oral compositions. During themanufacturing process, the activated or activated and conditioned yeastcells may be dried and stored for a period of time.

The biological or oral compositions of the invention are ingested by thesubject or used as an additive to be incorporated into food to beconsumed by the subject. Dietary supplement and nutritional compositionscomprising activated and conditioned yeast cells are encompassed by theinvention. Preferably, the subject is a human being.

In various embodiments, the biological or oral compositions of theinvention are used to produce a healthful benefit in a subject withuterine cancer or at high risk of developing uterine cancer. Inparticular, the biological composition of the invention can retard thegrowth of uterine cancer cells in an animal which received thecomposition orally. The composition can also be used to prolong the timeof survival of an animal with uterine cancer.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1 Activation and conditioning of yeast cells. 1 yeast cell culture;2 container; 3 electromagnetic field source; 4 electrode.

FIG. 2 Large scale propagation of yeast cells. 5 first container; 6second container; 7 third container; 8 yeast cell cultures; 9electromagnetic field source.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to biological compositions that canproduce a healthful benefit in a subject with uterine cancer. Thepresent invention provides methods for manufacturing the biologicalcompositions as well as methods for using the biological compositions.

In one embodiment, the invention provides biological compositions thatcomprise yeasts. Unlike the traditional use of yeasts in the making offood, the yeast cells of the invention are not used as a source ofenzymes that acts on the food ingredients. The yeasts are not a primarysource of nutrients for the subject. Nor are yeast cells used as acarrier, such as metal salts. The yeast cells of the invention are livewhen administered orally or ingested along with food by a subject.Without being bound by any theory or mechanism, the inventor believesthat the culture conditions activate and/or amplified the expression ofa gene or a set of genes in the yeast cells such that the yeast cellsbecomes highly effective in stimulating the animal's immune system,including both specific and non-specific immunological reactions, theresults of which are manifested as the overall healthful benefitsobserved in the treated subject. The healthful benefits provided byusing the biological compositions are demonstrated in animal models ofhuman uterine cancer which show inhibition of tumor growth and prolongedsurvival time of animals with the disease.

In another embodiment, the invention provides methods for making theyeast cells in the biological compositions. The starting materials arenormal yeast cells which can be readily obtained commercially or frompublic microorganism deposits. The methods of the invention comprise aset of culture conditions that can be applied reproducibly to activatethe yeast cells. The key feature of the culture conditions used in themethods of the invention is a series of alternating electromagneticfields of defined frequency ranges and field strengths which are appliedto the growing yeast cell culture. The method further comprises the stepof conditioning the activated live yeast cells to the acidic environmentof the stomach of the subject. The electromagnetic fields used in thesemethods can be created reproducibly at various scales, thus enablingeven the large scale manufacturing of the biological compositions of theinvention. By careful control of the culturing conditions, normal yeastcells can be activated routinely and reproducibly to become yeast cellsof the invention.

In yet another embodiment, the invention provides methods formanufacturing an oral composition comprising activated and conditionedyeasts of the invention, and additional ingredients, including but notlimited to pharmaceutically acceptable carriers or excipients, vitamins,herbs (including traditional Chinese medicine products), herbalextracts, minerals, amino acids, flavoring agents, coloring agents,and/or preservatives.

In yet another embodiment, the biological compositions can be added tofood which will be consumed by the subject. As known to those skilled inthe relevant art, many methods may be used to mix the biological or oralcompositions of the invention with food while the yeast cells remainviable. In a particular embodiment, the culture broth comprising liveyeast cells of the present invention are added directly to food justprior to consumption. Dried powders of the yeasts can also bereconstituted and added directly to food just prior to consumption.

In various embodiments, the oral compositions of the invention can beconsumed directly by a subject or be fed directly to a subject. Forexample, the subject may drink the culture broth or a fraction thereofthat comprises live activated and conditioned yeast cells. Oralcompositions comprising dried yeast cells can also be given as a soliddosage form to the subject.

Although it is not necessary, the biological or oral compositions of theinvention can be used in conjunction or in rotation with other types oftreatment modalities such as but not limited to surgery,chemotherapeutic agents, and radiation. Since the biologicalcompositions of the invention are administered orally, the assistance ofhealth professionals in administration of the composition is generallynot essential.

Described below in Section 5.1 are the yeast cells of the invention andmethods of their preparation. Section 5.2 describes the use of thebiological compositions of the invention in a subject suffering fromuterine cancer. The examples in Sections 6 to 9 demonstrate thetherapeutic benefits of an oral composition of the invention. Theactivated and conditioned yeast cells in the oral composition arecharacterized by their ability to (i) suppress the growth of cancercells in an animal model of human uterine cancer, or (ii) prolong thesurvival of animals with transplanted cancer cells in a model of humanuterine cancer, as compared to yeast cells which have not been activatedand conditioned.

5.1 PREPARATION OF THE YEAST CELL CULTURES

The yeast cells of the biological composition are produced by culturinga plurality of yeast cells in an appropriate culture medium in thepresence of an alternating electromagnetic field over a period of time.The method comprises a first step of activating the yeast cells and asecond step of conditioning the activated yeast cells. The activationprocess comprises culturing yeast cells in the presence of at least two,three, four or five electromagnetic fields of specific frequencies andfield strength. The conditioning process comprises further culturing ofthe activated yeast cells in a medium comprising plant extracts andextracts from the stomach of an animal, in the presence of at least oneelectromagnetic field. The activated and conditioned yeast cells can bestored as dried cells after drying the cells under appropriateconditions. The dried activated and conditioned yeast cells can be usedlater in large scale culturing processes for manufacturing thebiological compositions of the invention. The various culturingprocesses of the invention can be performed either as a batch process ora continuous process.

5.1.1 Yeasts

In various embodiments, yeasts of the genera of Saccharomyces, Candida,Crebrothecium, Geotrichum, Hansenula, Kloeckera, Lipomyces, Pichia,Rhodosporidium, Rhodotorula, Torulopsis, Trichosporon, and Wickerhamiacan be used in the invention. Generally, fungi used for foodmanufacturing are preferred.

Non-limiting examples of yeast strains include Saccharomyces sp.,AS2.311; Schizosaccharomyces pombe Linder, AS2.214, AS2.248, AS2.249,AS2.255, AS2.257, AS2.259, AS2.260, AS2.274, AS2.994, AS2.1043,AS2.1149, AS2.1178, IFFI 1056; Saccharomyces sake Yabe, ACCC2045;Saccharomyces uvarum Beijer, IFFI 1023, IFFI 1032, IFFI 1036, IFFI 1044,IFFI 1072, IFFI 1205, IFFI 1207; Saccharomyces rouxii Boutroux, AS2.178,AS2.180, AS2.370, AS2.371; Saccharomyces cerevisiae Hansen Var.ellipsoideus, ACCC2043, AS2.2, AS2.3, AS2.8, AS2.53, AS2.163, AS2.168,AS2.483, AS2.541, AS2.559, AS2.606, AS2.607, AS2.6 11, AS2.612;Saccharomyces carlsbergensis Hansen, AS2.116, AS2.162, AS2.189, AS2.200,AS2.216, AS2.265, AS2.377, AS2.417, AS2.420, AS2.440, AS2.441, AS2.443,AS2.444, AS2.459, AS2.595, AS2.605, AS2.638, AS2.742, AS2.745, AS2.748,AS2.1042; Rhodotorula aurantiaca (Saito)Ladder; AS2.102, AS2.107,AS2.278, AS2.499, AS2.694, AS2.703, AS2.704 and AS2.1146; Saccharomycescerevisiae Hansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037, ACCC2038,ACCC2039, ACCC2040, ACCC2041, ACCC2042, AS2.1, AS2.4, AS2.1 1, AS2.14,AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101, AS2.109,AS2.110, AS2.112, AS2.139, AS2.173, AS2.182, AS2.196, AS2.242, AS2.336,AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380, AS2.382, AS2.393,AS2.395, AS2.396, AS2.397, AS2.398, AS2.399, AS2.400, AS2.406, AS2.408,AS2.409, AS2.413, AS2.414, AS2.415, AS2.416, AS2.422, AS2.423, AS2.430,AS2.431, AS2.432, AS2.451, AS2.452, AS2.453, AS2.458, AS2.460, AS2.463,AS2.467, AS2.486, AS2.501, AS2.502, AS2.503, AS2.504, AS2.516, AS2.535,AS2.536, AS2.558, AS2.560, AS2.561, AS2.562, AS2.576, AS2.593, AS2.594,AS2.614, AS2.620, AS2.628, AS2.631, AS2.666, AS2.982, AS2.1190,AS2.1364, AS2.1396, IFFI 1001, IFFI 1002, IFFI 1005, IFFI 1006, IFFI1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI 1027, IFFI 1037,IFFI 1042, IFFI 1045, IFFI 1048, IFFI 1049, IFFI 1050, IFFI 1052, IFFI1059, IFFI 1060, IFFI 1062, IFFI 1202, IFFI 1203, IFFI 1209, IFFI 1210,IFFI 1211, IFFI 1212, IFFI 1213, IFFI 1215, IFFI 1221, IFFI 1224, IFFI1247, IFFI 1248, IFFI 1251, IFFI 1270, IFFI 1277, IFFI 1289, IFFI 1290,IFFI 1291, IFFI 1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI1302, IFFI 1307, IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311, IFFI 1331,IFFI 1335, IFFI 1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI1345, IFFI 1348, IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1441 and IFFI1443. Preferred yeast strains include but are not limited to S.cerevisiae AS2.501, AS2.502, AS2.503, AS2.504, AS2.535, AS2.558,AS2.560, AS2.561 and AS2.562.

Generally, yeast strains useful for the invention can be obtained fromprivate or public laboratory cultures, or publicly accessible culturedeposits, such as the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110–2209 and the China GeneralMicrobiological Culture Collection Center (CGMCC), China Committee forCulture Collection of Microorganisms, Institute of Microbiology, ChineseAcademy of Sciences, Haidian, P.O. Box 2714, Beijing, 100080, China.

Non-limiting examples of using yeast cells of the invention withSaccharomyces carlsbergensis Hansen strain AS2.605 are provided inSections 6 to 9 herein below. The yeast cells of the invention do notcomprise an enhanced level of selenium or chromium relative to thatfound in naturally occurring yeast cells. In certain embodiments, thebiological compositions do not comprise cells of Saccharomyces boulardii(for example, ATCC Accession No. 74366) or cells of a particular strainof Saccharomyces cerevisiae (strain Hansen CBS 5926) that is alsocommonly referred to as Saccharomyces boulardii.

Although it is preferred, the preparation of the yeast cells of theinvention is not limited to starting with a pure strain of yeast. Theyeast cells in the biological compositions may be produced by culturinga mixture of yeast cells of different species or strains. Theconstituents of a mixture of yeast cells can be determined by standardyeast identification techniques well known in the art.

In various embodiments of the invention, standard techniques forhandling, transferring and storing yeasts are used. Although it is notnecessary, sterile conditions or clean environments are highly desirablewhen carrying out the manufacturing processes of the invention,especially when the biological compositions are for human consumption.The manufacturing process can be adapted to meet regulatory guidelineson product safety and quality control by standard practice known in theart.

5.1.2 Electromagnetic Fields

As used herein, the terms “alternating electromagnetic field”,“electromagnetic field” or “EM field” are synonymous. An electromagneticfield useful in the invention can be generated by various means wellknown in the art. A schematic illustration of exemplary setups aredepicted respectively in FIG. 1. An electromagnetic field of a desiredfrequency and a desired field strength is generated by anelectromagnetic wave source (3) which comprises one or more signalgenerators that are capable of generating electromagnetic waves,preferably sinusoidal waves, and preferably in the frequency range of1,500 to 15,000 MHz and most preferably 10,100 to 12,700 MHz. Suchsignal generators are well known in the art. Signal generators capableof generating signal with a narrower frequency range can also be used.If desirable, a signal amplifier can also be used to increase the outputsignal, and thus the strength of the EM field.

The electromagnetic field can be applied to the culture by a variety ofmeans including placing the yeast cells in close proximity to a signalemitter connected to a source of electromagnetic waves. The signalgenerator is connected to the signal emitter by cables such as coaxialcables that can transmit signals up to greater than or equal to 30 GHz.Typically, the yeast cells are placed in a container which is made ofmaterial that is not an electric conductor, such as but not limited toplastic, resin, glass, and ceramic.

In one embodiment, the electromagnetic field is applied by signalemitters in the form of electrodes (4) that are submerged in a cultureof yeast cells (1). In a preferred embodiment, one of the electrodes isa metal plate which is placed on the bottom of a non-conductingcontainer (2), and the other electrode comprises a plurality of wires ortubes so configured inside the container such that the energy of theelectromagnetic field can be evenly distributed in the culture. Theelectrodes are preferably made of copper. For an upright culture vessel,the tips of the wires or tubes are placed within 3 to 30 cm from thebottom of the vessel (i.e., approximately 2% to 10% of the height of thevessel from the bottom). Table 1 provides exemplary set up for culturingthe yeast cells of the invention.

TABLE 1 height of culture medium in the distance electrodes are rangefor distance of non-conducting placed from the bottom the electrodesfrom container (cm) of the container (cm) the bottom (cm) 15 to 20 3 3to 5 20 to 30 5 5 to 7 30 to 50 7  7 to 10 50 to 70 10 10 to 15  70 to100 15 15 to 20 100 to 150 20 20 to 30 150 to 200 30 25 to 30

The number of electrodes used depends on both the volume of the cultureand the diameter of the electrode. For example, for a culture having avolume of 10 liter or less, two or three electrodes having a diameter ofbetween 0.5 to 2.0 mm can be used. For a culture volume of 10 to 100liter of culture, the electrodes can have a diameter of 3.0 to 5.0 mm.For a culture volume of 100 to 1,000 liter, the electrodes can have adiameter of 6.0 to 15.0 mm. For a culture having a volume greater than1,000 liter, the electrodes can have a diameter of between 20.0 to 25.0mm.

5.1.3 Activation of Yeast Cells

According to the invention, the method for producing activated yeastcells of the invention comprises culturing yeast cells in the presenceof at least two, three, four or five alternating electromagnetic (EM)fields.

The culture process can be initiated by inoculating 1,000 ml of mediumwith an inoculum of a selected yeast strain (such as one of thosedescribed in Section 5.1.1) such that the starting cell density of theculture is greater than about 10⁵ cells per ml. For example,Saccharomyces carlsbergensis Hansen strain AS2.605 can be used. Thestarting culture can be used to seed larger scale culture. The cultureis maintained initially at 28° C. to 32° C. for 22 to 30 hours prior toexposure to the EM field(s), typically at 30° C. for 28 hours.

The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.08mol/m³, preferably 0.04 mol/m³. The oxygen level can be controlled byany conventional means known in the art, including but not limited tostirring and/or bubbling.

The culture is most preferably carried out in a liquid medium whichcontains sources of nutrients assimilable by the yeast cells. Table 2provides an exemplary medium for culturing the yeast cells of theinvention.

TABLE 2 Medium Composition Quantity Sucrose or glucose 20 g Vitamin B₁₂60 μg Vitamin B₃ 60 μg Vitamin H 60 μg Vitamin B₆ 50 μg Fetal calf serum30 ml KH₂PO₄ 0.20 g MgSO₄.7H₂O 0.25 g NaCl 0.30 g CaSO₄.2H₂O 0.20 gCaCO₃.5H₂O 4.0 g Peptone 2.5 g Autoclaved water 1,000 ml

The culturing medium is heated to 45° C. and cooled before adding thevitamin B₁₂, vitamin B₃, vitamin H, vitamin B₆, and fetal calf serum.

In general, carbohydrates such as sugars, for example, sucrose, glucose,fructose, dextrose, maltose, xylose, and the like and starches, can beused either alone or in combination as sources of assimilable carbon inthe culture medium. The exact quantity of the carbohydrate source orsources utilized in the medium depends in part upon the otheringredients of the medium but, in general, the amount of carbohydrateusually varies between about 0.1% and 5% by weight of the medium and ispreferably between about 0.2% and 2%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium. Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are KH₂PO₄, (NH₄)₂HPO₄, CaCO₃,MgSO₄, NaCl, and CaSO₄.

It should be noted that the composition of the media provided in Table 2is not intended to be limiting. The process can be scaled up or downaccording to needs. Various modifications of the culture medium may bemade by those skilled in the art, in view of practical and economicconsiderations, such as the scale of culture and local supply of mediacomponents.

In certain embodiments, a series of at least two, three, four or five EMfields are applied to the culture of yeast cells, each having adifferent frequency within a stated range, and a different fieldstrength within a stated range. The EM fields can be applied in anyorder and by any means known in the art, such as the apparatus describedin Section 5.1.2. Although any of the following two, three or four EMfields can be applied, preferably, all five EM fields are applied.

For the first EM field, the frequency is in the range of 10,161 to10,170 MHz and the field strength is in the range of 220 to 240 mV/cm.The yeast culture is exposed to this first EM field at 30±2° C. forabout 24 hours.

For the second EM field, the frequency is in the range of 11,521 to11,530 MHz and the field strength is in the range of 210 to 230 mV/cm.The yeast culture is exposed to this second EM field at 30±2° C. forabout 8 hours.

For the third EM field, the frequency is in the range of 12,131 to12,140 MHz and the field strength is in the range of 255 to 275 mV/cm.The yeast culture is exposed to this third EM field at 30±2° C. forabout 24 hours.

For the fourth EM field, the frequency is in the range of 12,451 to12,460 MHz and the field strength is in the range of 260 to 280 mV/cm.The yeast culture is exposed to this fourth EM field at 30±2° C. forabout 8 hours.

For the fifth EM field, the frequency is in the range of 12,696 to12,705 MHz and the field strength is in the range of 270 to 290 mV/cm.The yeast culture is exposed to this fifth EM field at 30±2° C. forabout 24 hours.

In less preferred embodiments, the yeast cells can be cultured byexposure to two, three or four of the above-mentioned EM fields in adifferent order. The yeast cells can remain in the same container anduse the same set of electromagnetic wave generator and emitters whenswitching from one EM field to another EM field.

The cell density of the culture at the end of the activation process istypically greater than about 10⁶ to 10⁹ cells per ml (estimated byhematocytometer). The activated yeast cells may be recovered from theculture by various methods known in the art, and stored at a temperaturebelow about 0° C. to 4° C. The activated yeast cells recovered from theliquid culture may be dried and stored in powder form. Preferably, thepowder form of the yeast cells comprises greater than about 10⁷ to 10¹⁰yeast cells per gram.

5.1.4 Conditioning of Yeast Cells

According to the invention, performance of the activated yeast cells canbe optimized by culturing the activated yeast cells in the presence ofan extract from the stomach (e.g., the gastric juice) of an animal withphysiology similar to the subject to which the biological compositionwill be administered. The inclusion of this additional conditioningprocess allows the activated yeast cells to adapt to and endure theacidic environment of the subject's stomach. The method for conditioningactivated yeast cells of the invention comprises culturing yeast cellsin such materials in the presence of at least one EM field.

The culture process can be initiated by inoculating 1,000 ml of aconditioning medium with about 10 gram of dried activated yeastscontaining about 10¹⁰ cells per gram (as prepared by the methodsdescribed in Section 5.1.3). An equivalent number of yeast cells inculture, preferably greater than 10⁶ to 10⁹ cells per ml, morepreferably at 10⁸ cells per ml, can also be used as an inoculum. Theconditioning medium comprises per 1,000 ml about 700 ml of gastric juiceof an animal and about 300 ml of wild hawthorn juice. The process can bescaled up or down according to needs.

The gastric juice of an animal can be obtained from the stomach contentof a freshly slaughtered animal. Although not essential, the animal ispreferably kept under a clean environment, and fed a standard diet,preferably germ-free. For example, the content of the stomach of a120-day old pig is mixed with 2,000 ml of distilled water, and allowedto settle without stirring for 6 hours. The clear liquid above iscollected for use as the gastric juice used in the conditioning process.The gastric juice of a pig can be used to condition yeast cells for usein a variety of mammals, including humans. Other methods that can beused to collect the gastric juice include centrifugation or filtrationof the mixture to remove debris and/or microorganisms. The gastric juiceso obtained can be stored at 4° C. Preferably, the collection proceduresand storage are carried out under sterile conditions.

The wild hawthorn juice is an extract of wild hawthorn fruits preparedby slicing the fruits and drying the slices in air, preferably to lessthan 8% moisture (commercial dryer can be used if necessary), crushingthe dried fruits to less than 20 mesh, and mixing 1,500 ml of water per500 gram of the crushed wild hawthorn. The mixture is then allowed tosettle without stirring for 6 hours, and the clear liquid above iscollected for use as the wild hawthorn juice used in the conditioningprocess. Other methods that can be used to collect the hawthorn juiceinclude centrifugation or filtration of the mixture. Preferably, thecollection procedures and storage are carried out under sterileconditions.

The activated yeast cells are conditioned by culturing in at least oneof the following two EM fields which can be applied by the apparatusdescribed in Section 5.1.2 or any means known in the art:

The first EM field has a frequency in the range of 12,451 to 12,460 MHzand a field strength in the range of 280 to 300 mV/cm. The temperatureis maintained at 28° C. to 32° C., and typically at 30° C. The yeastculture is exposed to this first EM field for about 12 hours.

The second EM field has a frequency in the range of 12,696 to 12,705 MHzand a field strength in the range of 280 to 300 mV/cm. The temperatureis maintained at 28° C. to 32° C., and typically at 30° C. The yeastculture is exposed to this second EM field for about 34 hours.

In a preferred embodiment, the activated yeast cells are conditioned byculturing in both of the above-mentioned EM fields. In less preferredembodiments, the yeast cells are conditioned in the two different EMfields in a different order. In other embodiments, a series of EM fieldshaving field characteristics within the ranges stated above can beapplied to condition the yeast cells. The yeast cells can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The cell density of the culture at the end of the activation process istypically greater than about 10⁷ to 10¹⁰ cells per ml (estimated byhematocytometer). The activated and conditioned yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C.

The activated and conditioned yeast cells can be used directly in abiological composition or used as a starter culture for large scalemanufacturing. The activated and conditioned yeast cells recovered fromthe liquid culture may be dried and stored in powder form. Preferably,the powder form of the activated and conditioned yeast cells comprisesgreater than about 10⁸ to 10¹¹ yeast cells per gram.

5.1.5 Large Scale Manufacturing

The present invention also encompasses methods of manufacturing of thebiological compositions of the invention at a large scale. The activatedand conditioned yeast cells as prepared by Sections 5.1.3 and 5.1.4 arepropagated on a large scale to make the biological compositions of theinvention. The method comprises culturing the yeast cells in thepresence of one or more EM fields for a period of time, diluting thegrowing yeast cells with fresh medium, and repeating the process. Themethod can be carried out as a batch process or a continuous process.

In one preferred embodiment, a set of three containers (5, 6, 7) eachcomprising a set of electrodes for generating an electromagnetic fieldas described in Section 5.1.2 are set up each with 1,000 liters of aculture medium. See FIG. 2. The culture medium comprises nutrientsassimilable by the yeast cells as shown in Table 3.

TABLE 3 Material Quantity Wild hawthorn juice 300 liters Jujube juice300 liters Wu wei zi juice 300 liters Soybean juice 100 liters

The wild hawthorn juice is an extract of fresh wild hawthorn fruitsprepared by washing the fruits clean, drying the fruits in air or usinga commercial dryer to less than 8% moisture, crushing the dried fruitsto less than 20 mesh, and mixing the crushed wild hawthorn with water ata ratio of 400 liters of water per 100 kg of crushed fruits. The mixtureis then stirred continuously for 12 hours while the temperature ismaintained at 28° C. to 30° C. The mixture is then centrifuged at 1,000rpm to collect the supernatant which is used as described above.Preferably, the procedures are carried out under sterile conditions.

The jujube juice is an extract of fresh jujube fruits prepared bywashing the fruits clean, drying the fruits to less than 8% moisture,crushing the dried fruits to less than 20 mesh, and mixing the crushedjujube with water at a ratio of 400 liters of water per 100 kg ofcrushed fruits. The mixture is then stirred continuously for 12 hourswhile the temperature is maintained at 28° C. to 30° C. The mixture isthen centrifuged at 1,000 rpm to collect the supernatant which is usedas described above. Preferably, the procedures are carried out understerile conditions.

The wu wei zi juice is an extract of fresh berries of Schisandrachinensis plant prepared by washing the berries, drying the fruits toless than 8% moisture, crushing the dried berries to less than 20 mesh,and mixing the crushed berries with water at a ratio of 400 liters ofwater per 100 kg of crushed berries. The mixture is then stirredcontinuously for 12 hours while the temperature is maintained at 28° C.to 30° C. The mixture is then centrifuged at 1,000 rpm to collect thesupernatant which is used as described above. Preferably, the proceduresare carried out under sterile conditions.

The soybean juice is prepared by washing the soybeans, drying thesoybeans to less than 8% moisture, crushing the soybeans to less than 20mesh, and mixing the crushed soybeans with water. For 30 kg of soybeans,130 liters of water is used. The mixture is then stirred continuouslyfor 12 hours while the temperature is maintained at 28° C. to 30° C. Themixture is then centrifuged at 1,000 rpm to collect the supernatantwhich is used as described above. Preferably, the procedures are carriedout under sterile conditions.

The first container is inoculated with activated or activated andconditioned yeast cells as prepared by the methods of Sections 5.1.4 and5.1.5. About 1,000 gram of dried yeast powder are added to 1,000 literof culture medium. Each gram of the dried yeast powder comprises about10¹⁰ yeast cells. Instead of dried yeast cells, an equivalent number ofyeast cells in a liquid medium can also be used, preferably greater thanabout 10⁶ to 10⁹ cells per ml, more preferably about 10⁷ cells per ml.

The yeast cells in the first container (5) are then subjected to aseries of two EM fields. For the first EM field, which can be applied bythe apparatus described in Section 5.1.2, the frequency is in the rangeof 12,451 to 12,460 MHz and the field strength is in the range of 300 to320 mV/cm. The yeast culture is exposed to this first EM field for about8 hours. The yeast cells are then subjected to a second EM field havinga frequency in the range of 12,696 to 12,705 MHz and a field strength inthe range of 320 to 340 mV/cm. The yeast culture is exposed to thissecond EM field for about 12 hours. The yeast cells from the firstcontainer are then transferred to the second container which containsabout 1,000 liter of the culture medium. In effect, the first yeastculture is diluted by about 50% with fresh culture medium.

In the second container (6), the yeast cells are again subjected to aseries of two EM fields. The frequencies used in the second containerare similar to those used in the first container but the field strengthsare marginally lower. The first EM field has a frequency in the range of12,451 to 12,460 MHz and a field strength in the range of 320 to 340mV/cm. The yeast culture is exposed to this EM field for about 8 hours.The yeast cells are then subjected to a second EM field having afrequency in the range of 12,696 to 12,705 MHz and a field strength inthe range of 330 to 350 mV/cm. The yeast culture is exposed to thissecond EM field for about 12 hours. The yeast cells from the secondcontainer are then transferred to the third container which contains yetanother 1,000 liter of the culture medium. Again, the second yeastculture is diluted by about 50% with fresh culture medium.

In the third container (7), the yeast cells are again subjected to aseries of two EM fields. The frequencies used in the third container aresimilar to those used in the first and second container but the fieldstrengths are lower. The first EM field has a frequency in the range of12,451 to 12,460 MHz and field strength in the range of 250 to 270mV/cm. The yeast culture is exposed to this EM field for about 12 hours.The yeast cells are then subjected to a second EM field having afrequency in the range of 12,696 to 12,705 MHz and a field strength inthe range of 250 to 270 mV/cm. The yeast culture is exposed to this EMfield for about 24 hours.

The yeast cell culture resulting from the end of this stage can be useddirectly as an oral composition of the invention, or used to form othercompositions encompassed by the invention.

The cell density of the culture at the end of the large scalemanufacturing process is typically greater than about 10⁸ to 10¹⁰ cellsper ml (estimated by hematocytometer). The concentration of yeast cellsin the medium can be concentrated or diluted accordingly. In certainembodiments, the concentration of yeast cells in the medium is in therange of 10³ to 10¹⁰ cells per ml. In less preferred embodiments, theconcentration of yeast cells in the medium is in the range of 10³ to 10⁶cells per ml. In more preferred embodiments, the concentration of yeastcells in the medium is greater than 10⁶ to 10¹⁰ cells per ml. In mostpreferred embodiments, the concentration of yeast cells in the medium isin the range of 10⁶ to 5×10⁸ cells per ml.

Other ingredients that enhance the healthful benefits, pharmacologicalproperties and/or organoleptic characteristics of the composition can beadded to the yeast cell culture. To maintain viability and freshness ofthe composition, it is preferred that the various downstream andpackaging process be carried out below room temperature, and preferablyat 0° C. to 4° C. In one embodiment, the yeast cell culture can bepackaged in liquid containers.

In another embodiment, the activated and conditioned yeast cells can bedried as follows. The yeast cell culture is first centrifuged under 75to 100 g for 10 to 20 minutes to remove the supernatant. The residuewhich may contain up to 85% moisture is dried in a first dryer at atemperature not exceeding 60±2° C. for a period of 5 minutes so thatyeast cells quickly became dormant. The yeast cells were then sent to asecond dryer and dried at a temperature not exceeding 65±2° C. for aperiod of about 8 minutes to further remove at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99% ofwater. For example, the yeast cells may be dried to remove at least 88%of water so the dried yeast cells may contain up to 12% moisture.

After cooling to room temperature, the dried yeast cells can be packagedby standard pharmaceutical methods in various solid dosage form, eachcontaining a predetermined amount of the dried material. In a preferredembodiment, the dried material comprises about 10⁵ to 10¹¹ cells pergram. In a more preferred embodiment, the dried material comprises about10⁸ to 5×10¹⁰ cells per gram. In a most preferred embodiment, the driedmaterial comprises about 5×10⁸ cells per gram.

In general, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers.

5.1.6 Preferred Embodiments

In one preferred embodiment, the invention provides a method forpreparing a biological composition comprising activated and conditionedyeast cells, said method comprising in any order the steps of:

-   (a) culturing the yeast cells in a first electromagnetic field    having a frequency at 10,162 MHz and a field strength of 235 mV/cm;-   (b) culturing the yeast cells in a second electromagnetic field    having a frequency at 11,526 MHz and a field strength of 226 mV/cm;-   (c) culturing the yeast cells in a third electromagnetic field    having a frequency at 12,132 MHz and a field strength of 266 mV/cm;-   (d) culturing the yeast cells in a fourth electromagnetic field    having a frequency at 12,458 MHz and a field strength of 265 mV/cm;    and-   (e) culturing the yeast cells in a fifth electromagnetic field    having a frequency at 12,698 MHz and a field strength of 275 mV/cm;    and after the last of the first five steps, the following steps in    any order:    -   (f) culturing the yeast cells in a liquid medium comprising wild        hawthorn juice and gastric juice of a mammal in a sixth        electromagnetic field having a frequency at 12,458 MHz and a        field strength of 284 mV/cm; and    -   (g) culturing the yeast cells in a liquid medium comprising wild        hawthorn juice and gastric juice of a mammal in a seventh        electromagnetic field having a frequency at 12,698 MHz and a        field strength of 297 mV/cm.

The activated and conditioned yeast cells obtained at the conclusion ofthis method is encompassed by the invention. Preferably, the yeast cellsare Saccharomyces carlsbergensis Hansen strain AS2.605. These yeastcells can be used in the following method of further expanding number ofactivated and conditioned yeast cells.

In another preferred embodiment, the invention provides a method of massproducing a biological composition comprising activated and conditionedyeast cells, said method comprising culturing the activated andconditioned yeast cells prepared by the preferred embodiment describedabove in this section, in a medium comprising wild hawthorn juice,jujube juice, wu wei zi juice, and soybean juice, and in the presence ofone or more series of electromagnetic fields. Each series of EM fieldscomprises two EM fields in the order stated:

-   (h) an eighth electromagnetic field or series of electromagnetic    fields having a frequency at 12,458 MHz and a field strength in the    range of 250 to 340 mV/cm, preferably at three fields strengths,    e.g., in the order of 316 mV/cm, 328 mV/cm, and 262 mV/cm; and-   (i) a ninth electromagnetic field or series of electromagnetic    fields having a frequency at 12,698 MHz and a field strength in the    range of 250 to 350 mV/cm, preferably at three fields strengths,    e.g., in the order of 327 mV/cm, 346 mV/cm, and 262 mV/cm.

The series may be repeated several times, such as three times, each timeusing a slightly lower field strength.

5.2 METHOD OF USES

5.2.1 Uses in Subjects with Uterine Cancer

The present invention further provides methods of use of the biologicalcompositions of the invention. In one embodiment, the biologicalcomposition is used as a medicament for treatment of uterine cancer. Inanother embodiment, the biological composition is used as a dietarysupplement, health food, or health drink. The methods compriseadministering an effective amount of the biological composition to asubject in need. The biological composition may be administered orally,in liquid or solid form, or enterally through a feeding tube. As usedherein, the term “an effective amount” means an amount sufficient toprovide a therapeutic or healthful benefit in the context of uterinecancer.

According to the invention, the biological composition can produce ahealthful benefit in a subject suffering from uterine cancer.Preferably, the subject is a human being. The subject in need is one whois diagnosed with uterine cancer, with or without metastasis, at anystage of the disease (e.g., TX, T0, Tis, T1, T2, T3, T4, NX, N0, N1, MX,M0 and M1). As used herein, the term “uterine cancer” includes but isnot limited to squamous cell carcinoma, endometrioid carcinoma,adenoacanthoma, adenosquamous carcinoma, papillary serous carcinomas,and clear cell adenocarcinomas.

The subject may be a uterine cancer patient who is receivingconcurrently other treatment modalities against the uterine cancer. Thesubject can be a uterine cancer patient who had undergone a regimen oftreatment (e.g., chemotherapy and/or radiation) and whose cancer isregressing. The subject may be a uterine cancer patient who hadundergone a regimen of treatment (e.g., surgery) and who appears to beclinically free of the uterine cancer. The biological composition of theinvention can be administered adjunctively with any of the treatmentmodalities, such as but not limited to chemotherapy, radiation, and/orsurgery. For example, the biological composition can be used incombination with one or more chemotherapeutic or immunotherapeuticagents, such as cyclosporin A, Taxol, Cisplatin, Carboplatin,Adriamycin, Doxil, and Topotecan. The biological composition can also beused after other regimen(s) of treatment is concluded.

The subject may be one who has not yet been diagnosed with uterinecancer but are predisposed to or at high risk of developing uterinecancer as a result of genetic factors and/or environmental factors. Thesubject may also be one who displays characteristics that are associatedwith a high risk of uterine cancer, such as nodules detected by computertomographic scanning or suspect cells in biopsy and/or body fluids.

Depending on the subject, the therapeutic and healthful benefits rangefrom inhibiting or retarding the growth of the uterine cancer and/or thespread of the uterine cancer to other parts of the body (i.e.,metastasis), palliating the symptoms of the cancer, improving theprobability of survival of the subject with the cancer, prolonging thelife expectancy of the subject, improving the quality of life of thesubject, and/or reducing the probability of relapse after a successfulcourse of treatment (e.g., surgery, chemotherapy or radiation). Thesymptoms associated with uterine cancer include abnormal vaginal and/oruterine spotting or bleeding, such as between periods or afterintercourse, white or clear vaginal discharge, difficult or painfulurination, discomfort during intercourse, pain or cramping in the pelvicarea, and discomfort during intercourse.

In particular, the invention provides a method for retarding the growthof uterine cancer cells in a subject, such as a human, comprisingadministering orally to the subject a biological composition of theinvention. The invention also provide a method for prolonging the timeof survival of a subject inflicted with uterine cancer, preferably ahuman patient, comprising administering orally to the subject abiological composition of the invention.

The effective dose will vary with the subject treated. The effectivedose for the subject will also vary with the condition to be treated andthe severity of the condition to be treated. The dose, and perhaps thedose frequency, will also vary according to the age, body weight, andresponse of the individual subject. In general, the total daily doserange of activated and conditioned yeast cells for a subject inflictedwith uterine cancer is from about 10⁵ to 10¹¹ cells per day; preferably,about 10⁸ to 5×10¹⁰ cells per day; more preferably, about 2×10⁹ cellsper day in powder form or 9×10⁸ to 1×10¹⁰ cells per day in liquidpreparations, administered in single or divided doses orally. The lengthof time for a course of treatment should be at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 7weeks, at least 10 weeks, at least 13 weeks, at least 15 weeks, at least20 weeks, at least 6 months, or at least 1 year. It may be necessary touse dosages outside these ranges in some cases as will be apparent tothose skilled in the art. In certain embodiments, the oral compositionscan be administered for a period of time until the symptoms and/orinfection of the patients by the bacteria and viruses are under control,or when the disease has regressed partially or completely. For use as adietary supplement, the total daily dose range should be from about 10⁵to 10¹¹ cells per day; preferably, about 5×10⁷ to 5×10⁹ cells per day.The oral compositions can be administered as a dietary supplement for aslong as 6 months, or in accordance with recommended length of use underthe Dietary Supplement Health and Education Act (DSHEA) or othergovernment or industry guidelines. Further, it is noted that thenutritionist, dietician, clinician or treating physician will know howand when to interrupt, adjust, or terminate use of the biologicalcomposition as a medicament or dietary supplement in conjunction withindividual patient response.

The effect of the biological compositions of the invention ondevelopment and progression of uterine cancer can be monitored by anymethods known to one skilled in the art, including but not limited tomeasuring: a) changes in the size and morphology of the tumor usingimaging techniques such as a computed tomographic (CT) scan or asonogram; and b) changes in levels of biological markers of risk foruterine cancer.

5.2.2 Formulations

The biological compositions of the present invention comprise activatedand conditioned live yeast cells prepared as described above in Section5.1, as active ingredient, and can optionally contain a pharmaceuticallyacceptable carrier or excipient, and/or other ingredients provided thatthese ingredients do not kill or inhibit the yeast cells. Otheringredients that can be incorporated into the biological compositions ofthe present invention, may include, but are not limited to, herbs(including traditional Chinese medicine products), herbal extracts,vitamins, amino acids, metal salts, metal chelates, coloring agents,flavor enhancers, preservatives, and the like.

Any dosage form may be employed for providing the subject with aneffective dosage of the oral composition. Dosage forms include tablets,capsules, dispersions, suspensions, solutions, and the like. In oneembodiment, compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, or tablets, each containing a predetermined amount of activatedand conditioned yeast cells, as a powder or granules or as a solution ora suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. Such products can be used as pharmaceuticals or dietarysupplements, depending on the dosage and circumstances of its use.

The oral compositions of the present invention may additionally includebinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); binders or fillers (e.g., lactose,pentosan, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets or capsules can be coated bymethods well known in the art.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. The temperature of the liquid used to reconstitute the driedproduct should be less than 65° C. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). As describedbelow, the preparations can also be made to resemble foods or beverages,containing buffer salts, flavoring, coloring and sweetening agents asappropriate. In certain embodiments, the oral composition is a cellsuspension comprising about 10³ to 10¹⁰ cells per ml. The oralcomposition can be produced by diluting or concentrating the yeastculture medium produced by the method of Section 5.1.5 as required. Inless preferred embodiments, the oral composition is a cell suspensioncontaining about 10³ to 10⁶ cells per ml. In more preferred embodiments,the oral composition is a cell suspension containing greater than about10⁶ to 10¹⁰ cells per ml. In most preferred embodiments, the oralcomposition is a cell suspension containing about 10⁶ to 5×10⁸ cells perml. The oral composition can be formulated as a health drink andpackaged in liquid containers, each containing a predetermined amount ofthe liquid yeast culture. Standard methods of quality control andpackaging are applied to produce in one embodiment of the invention,oral compositions packaged in liquid containers each comprising about 1ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml, 75ml, 100 ml, 150 ml, 200 ml, 250 ml, 500 ml, 750 ml, or 1,000 ml of thelive yeast cells. The number of container to be taken each day to obtainthe total daily dose in a subject depends on the number of activated andconditioned yeast cells contained within each container. For example, acontainer may comprise 50 ml of liquid with 107 cells per ml and when atotal daily dose of about 2×10⁹ cells per day is desired, a subject candrink 4 containers per day to obtain the desired total daily dose.

Generally, because of their ease of administration, tablets and capsulesrepresent the most advantageous oral dosage unit form, in which casesolid pharmaceutical carriers as described above are employed. In apreferred embodiment, the composition is a capsule. The capsules can beformulated by any commercially available methods. In certainembodiments, the composition is a capsule containing 5 mg, 10 mg, 15 mg,20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1.0 gram, 1.25 gram,1.5 gram, or 2.0 gram of live yeast cells in powder form. The powder inthe capsule comprises about 10⁵ to about 10¹¹ cells per gram; morepreferably, about 10⁸ to 5×10¹⁰ cells per gram; and most preferably,about 5×10⁸ cells per gram. The number of capsule to be taken each dayto obtain the total daily dose in a subject depends on the number ofactivated and conditioned yeast cells contained within each capsule. Forexample, a capsule may comprise about 500 mg of powder with 5×10⁸ cellsper gram. To achieve a total daily dose of about 2×10⁹ cells per day, asubject can take two capsules at a time for four times per day.

In another embodiment, the biological compositions comprising activatedand conditioned yeast cells can be added directly to foods so that aneffective amount of yeast cells is ingested during normal meals. Anymethods known to those skilled in the art may be used to add to orincorporate the biological compositions into natural or processed foods,provided that the activated and conditioned yeast cells remain viable.Preferably, the nutritional compositions of the invention are made andstored under conditions, such as temperature, from about 0° C. to 4° C.As used herein, the term “food” broadly refers to any kind of material,liquid or solid, that is used for nourishing an animal, and forsustaining normal or accelerated growth of an animal including humans.Many types of food products or beverages, such as but not limited to,fruit juice, herbal extracts, tea-based beverages, dairy products,soybean product (e.g., tofu), and rice products, can be used to formnutritional compositions comprising the activated and conditioned yeastcells of the invention.

The invention is further defined by reference to the following exampledescribing in detail the animal trials conducted to study the efficacyand safety of activated and conditioned yeast cells of the invention.

6. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a 615 strain mouse model of humanuterine cancer. The growth of the tumor in the mice was studied.

Murine transplantable uterine cancer type CRL-1622 closely resembles thehuman uterine cancer pathology. The uterine cancer cell line CRL-1622can be ordered from the American Type Culture Collection.

The biological composition comprising 10⁸ cells per ml of activated andconditioned yeast cells of the strain Saccharomyces carlsbergensisHansen strain AS2.605 was prepared by the methods described in Section5.1 and subsections therein.

6.1 Animal Preparation

The animals used to generate the uterine cancer cells for theexperiments were female 615 mice (obtainable from the Chinese Academy ofMilitary Medical Sciences, Beijing, China), 4 to 6 months, having anaverage body weight of about 20 to 22 gram. About 1.2×10⁷ viable tumorcells of the uterine cancer cell line CRL-1 622 (obtainable from theCancer Institute, Chinese Academy of Medical Sciences, Beijing, China)in about 0.2 ml culture suspension were injected subcutaneously into theanimals.

6.2 Experimental Design

The mice injected with tumor cells were immediately divided into 4experimental groups of ten mice per group and one control group. Thefour experimental groups were triplicated (i.e., using a total of 120mice in the experimental groups). In group AY, the mice received 0.3 mlof the biological composition once per day. In group NY, the micereceived 0.3 ml of the untreated yeast cells once per day. In group MMC,the mice were injected subcutaneously with 0.8 mg of mitomycin C (MMC)per kg body weight per day. In group CK1, the mice received 0.3 ml ofphysiological saline once per day. A fifth group of mice, group CK2,which did not receive tumor cells, was given 0.3 ml of physiologicalsaline per day.

The mice received the biological composition, untreated yeast cells, MMCor saline on the same day as the tumor cells were transplanted. The micein group CK2 also started receiving saline on the same day as the otherfour groups. The biological composition, untreated yeast cells andsaline were administered orally by a feeding tube and the MMC bysubcutaneous injection for 30 consecutive days. On the ₃₁st day fromtumor inoculation, the mice were sacrificed. The weight of the mice andthe weight of the tumor were determined by standard techniques.

6.3 Results

Table 4 shows the differences in the weight of the mice and tumors ofthe mice in the various treatment and control groups.

TABLE 4 mean weight of tumor mean weight of mice and nodules andstandard Group standard deviation (g) deviation (mg) AY 18.9 ± 1.7 1.56± 0.54 NY 17.6 ± 2.3 4.43 ± 2.45 MMC 18.3 ± 3.1 3.56 ± 2.32 CK1 17.4 ±3.7 4.82 ± 2.46 CK2 19.6 ± 2.6 not applicable

The mice bearing uterine cancer cells that received 0.3 ml of thebiological composition of the invention (group AY) showed the leastdeviation in body weight as compared to healthy mice not injected withtumor cells (group CK2). The mice in group AY also had less tumor massas compared to mice that did not receive treatment (group CK1) as wellas the mice in group NY (0.3 ml of untreated yeast cells per day) andthe mice in group MMC (0.8 mg of mitomycin C per kg body weight perday).

7. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a model of human uterine cancer in a 615strain mouse. The survival time of mice after tumor injection andtreatment was studied.

The biological composition comprising 10⁸ cells per ml of activated andconditioned yeast cells of the strain Saccharomyces carlsbergensisHansen strain AS2.605 was prepared by the methods described in Section5.1 and subsections therein.

7.1 Animal Preparation

The animals were prepared in a similar manner as described in Section6.1.

7.2 Experimental Design

The mice injected with tumor cells were immediately divided into 4experimental groups of ten mice per group and one control group. Thefour experimental groups were triplicated (i.e., using a total of 120mice in the experimental groups). In group 2AY, the mice received 0.5 mlof the biological composition once per day. In group 2NY, the micereceived 0.5 ml of the untreated yeast cells once per day. In group2MMC, the mice were injected subcutaneously with 1.2 mg of mitomycin C(MMC) per kg body weight per day. In group 2CK1, the mice received 0.5ml of physiological saline once per day. A fifth group of mice, group2CK2, which did not receive tumor cells, was given 0.5 ml ofphysiological saline per day.

The mice received the biological composition, untreated yeast cells, MMCor saline on the same day as the tumor cells were transplanted. The micein group 2CK2 also started receiving saline on the same day as the otherfour groups. The biological composition, untreated yeast cells andsaline were administered orally by a feeding tube and the MMC bysubcutaneous injection for 30 consecutive days. The mice were observedover 6 months from the day of tumor inoculation and survival wasrecorded. The weight of the mice and the weight of the tumor weredetermined by standard techniques.

7.3 Results

Table 5 shows the number of mice in the various treatment and controlgroup that survived the tumor injection over a period of 6 months. Eachof the 30 mice in each group received 30 consecutive days of eitheruntreated yeast cells, MMC, saline or a biological composition of theinvention. Table 6 shows the weight of the mice that survived and theweight of their tumors in the various treatment and control groups.

TABLE 5 Number of live animals remaining in the groups after 30 days oftreatment Time after cessation of Group Group Group Group Grouptreatment 2AY 2NY 2MMC 2CK1 2CK2 0 month 30 30 30 30 30 1 month 30 21 2823 30 2 months 30 11 26 12 30 3 months 30 2 18 3 30 4 months 30 0 7 0 305 months 30 0 0 0 30 6 months 30 0 0 0 30

TABLE 6 mean weight of mice mean weight of tumor and standard nodulesand standard Group deviation (g) deviation (mg) 2AY 20.2 ± 2.5 78.7 ±17.4 2NY all animals dead all animals dead 2MMC all animals dead allanimals dead 2CK1 all animals dead all animals dead 2CK2 21.4 ± 2.6 notapplicable

All of the mice bearing uterine cancer cells that received 0.5 ml of thebiological composition of the invention (group 2AY) survived for morethan 6 months and the tumor never reoccurred. On the contrary, the micein group 2NY (0.5 ml of untreated yeast cells per day), group 2MMC (1.2mg of mitomycin C per kg body weight per day) and group 2CK1 (0.5 ml ofsaline per day) all died during the experiment.

As in Example 6, the mice bearing uterine cancer cells that received 0.5ml of the biological composition of the invention (group 2AY) showed theleast deviation in the weight of mice as compared to healthy mice notinjected with tumor cells (group 2CK2).

8. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a kun ming mouse model of human uterinecancer. The growth of the tumor in the mice was studied.

Murine transplantable uterine cancer type HTB-114 closely resembles thehuman uterine cancer pathology. The uterine cancer cell line HTB-114 canbe ordered from the American Type Culture Collection.

The biological composition comprising 10⁸ cells per ml of activated andconditioned yeast cells of the strain Saccharomyces carlsbergensisHansen strain AS2.605 was prepared by the methods described in Section5.1 and subsections therein.

8.1 Animal Preparation

The animals used to generate the uterine cancer cells for theexperiments were female kun ming mouse (obtainable from the ChineseAcademy of Military Medical Sciences, Beijing, China), 5 to 6 months,having an average body weight of about 20 to 22 gram. About 1.2×10⁷viable tumor cells of the uterine cancer cell line HTB-114 (obtainablefrom the Cancer Institute, Chinese Academy of Medical Sciences, Beijing,China) in about 0.2 ml culture suspension were injected subcutaneouslyinto the animals.

8.2 Experimental Design

The mice injected with tumor cells were immediately divided into 4experimental groups of ten mice per group and one control group. Thefour experimental groups were triplicated (i.e., using a total of 120mice in the experimental groups). In group AY, the mice received 0.3 mlof the biological composition once per day. In group NY, the micereceived 0.3 ml of the untreated yeast cells once per day. In group VCR,the mice were injected intravenously with 3 mg of vinblastine (VCR) perkg body weight per day. In group CK1, the mice received 0.3 ml ofphysiological saline once per day. A fifth group of mice, group CK2,which did not receive tumor cells, was given 0.3 ml of physiologicalsaline per day.

The mice received the biological composition, untreated yeast cells, VCRor saline on the same day as the tumor cells were transplanted. The micein group CK2 also started receiving saline on the same day as the otherfour groups. The biological composition, untreated yeast cells andsaline were administered orally by a feeding tube and the VCR byintravenous injection for 30 consecutive days. On the 31^(st) day fromtumor inoculation, the mice were sacrificed. The weight of the mice andthe weight of the tumor were determined by standard techniques.

8.3 Results

Table 7 shows the differences in the body weight and the weight of tumorof the mice in the various treatment and control groups.

TABLE 7 mean weight of tumor mean weight of mice and nodules andstandard Group standard deviation (g) deviation (g) AY 19.6 ± 2.6 1.65 ±0.67 NY 17.7 ± 2.7 4.33 ± 2.34 VCR 18.3 ± 3.3 4.36 ± 2.45 CK1 18.4 ± 3.44.32 ± 2.36 CK2 20.5 ± 2.5 not applicable

The mice bearing uterine cancer cells that received 0.3 ml of thebiological composition of the invention (group AY) showed the leastdeviation in the weight of mice as compared to healthy mice not injectedtumor cells (group CK2). The mice in group AY also had less tumor massas compared to mice that did not receive treatment (group CK1) as wellas the mice in group NY (0.3 ml of untreated yeast cells per day) andthe mice in group VCR (3 mg of vinblastine per kg body weight per day).

9. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a kun ming mouse model of human uterinecancer. The survival time of mice after tumor injection and treatmentwas studied.

The biological composition comprising 10⁸ cells per ml of activated andconditioned yeast cells of the strain Saccharomyces carlsbergensisHansen strain AS2.605 was prepared by the methods described in Section5.1 and subsections therein.

9.1 Animal Preparation

The animals were prepared in a similar manner as described in Section8.1.

9.2 Experimental Design

The mice injected with tumor cells were immediately divided into 4experimental groups of ten mice per group and one control group. Thefour experimental groups were triplicated (i.e., using a total of 120mice in the experimental groups). In group 2AY, the mice received 0.5 mlof the biological composition once per day. In group 2NY, the micereceived 0.5 ml of the untreated yeast cells once per day. In group2VCR, the mice were injected intravenously with 4.5 mg of vinblastine(VCR) per kg body weight per day. In group 2CK1, the mice received 0.5ml of physiological saline once per day. A fifth group of mice, group2CK2, which did not receive tumor cells, was given 0.5 ml ofphysiological saline per day.

The mice received the biological composition, untreated yeast cells, VCRor saline on the same day as the tumor cells were transplanted. The micein group 2CK2 also started receiving saline on the same day as the otherfour groups. The biological composition, untreated yeast cells andsaline were administered orally by a feeding tube and the VCR byintravenous injection for 30 consecutive days. The mice were observedover 6 months from the day of tumor inoculation and survival wasrecorded. The weight of the mice and the weight of the tumor weredetermined by standard techniques.

9.3 Results

Table 8 shows the number of mice in the various treatment and controlgroup that survived the tumor injection over a period of 6 months. Eachof the 30 mice in each group received 30 consecutive days of eitheruntreated yeast cells, VCR, saline or a biological composition of theinvention. Table 9 shows the weight of the mice that survived and theweight of their tumors in the various treatment and control groups.

TABLE 8 Number of live animals remaining in the groups after 30 days oftreatment Time after cessation of Group Group Group Group Grouptreatment 2AY 2NY 2VCR 2CK1 2CK2 0 month 30 30 30 30 30 1 month 30 30 2821 30 2 months 30 16 19 17 30 3 months 30 3 11 5 30 4 months 30 0 3 0 305 months 30 0 0 0 30 6 months 30 0 0 0 30

TABLE 9 mean weight of tumor mean weight of mice and nodules andstandard Group standard deviation (g) deviation (mg) 2AY 21.6 ± 2.7103.5 ± 17.8 2NY all animals dead all animals dead 2VCR all animals deadall animals dead 2CK1 all animals dead all animals dead 2CK2 22.7 ± 2.4not applicable

The mice bearing uterine cancer cells that received 0.5 ml of thebiological composition of the invention (group 2AY) survived for morethan 6 months and the tumor never reoccurred. On the contrary, the micein group 2NY (0.5 ml of untreated yeast cells per day), group 2VCR (4.5mg of vinblastine per kg body weight per day) and group 2CK1 (0.5 ml ofsaline per day) all died during the experiment.

As in Example 8, the mice bearing uterine cancer cells that received 0.5ml of the biological composition of the invention (group 2AY) showed theleast deviation in the weight of mice as compared to healthy mice notinjected with tumor cells (group 2CK2).

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1. A method of treating yeast cells, comprising at least two stepsselected from the group consisting of: a culturing yeast cells in afirst electromagnetic field having a frequency in the range of 10,161 to10,170 MHz and a field strength in the range of 220 to 240 mV/cm; bculturing the yeast cells in a second electromagnetic field having afrequency in the range of 11,521 to 11,530 MHz and a field strength inthe range of 210 to 230 mV/cm; c culturing the yeast cells in a thirdelectromagnetic field having a frequency in the range of 12,131 to12,140 MHz and a field strength in the range of 255 to 275 mV/cm; dculturing the yeast cells in a fourth electromagnetic field having afrequency in the range of 12,451 to 12,460 MHz and a field strength inthe range of 260 to 280 mV/cm; and e culturing the yeast cells in afifth electromagnetic field having a frequency in the range of 12,696 to12,705 MHz and a field strength in the range of 270 to 290 mV/cm.
 2. Amethod of treating yeast cells, comprising at least two steps selectedfrom the group consisting of: a culturing yeast cells in a firstelectromagnetic field having a frequency in the range of 10,161 to10,170 MIHz and a field strength in the range of 220 to 240 mV/cm; bculturing the yeast cells in a second electromagnetic field having afrequency in the range of 11,521 to 11,530 MIIIz and a field strength inthe range of 210 to 230 mV/cm; c culturing the yeast cells in a thirdelectromagnetic field having a frequency in the range of 12,131 to12,140 MHz and a field strength in the range of 255 to 275 mV/cm; dculturing the yeast cells in a fourth electromagnetic field having afrequency in the range of 12,451 to 12,460 MIHz and a field strength inthe range of 260 to 280 mV/cm; and e culturing the yeast cells in afifth electromagnetic field having a frequency in the range of 12,696 to12,705 MHz and a field strength in the range of 270 to 290 mV/cm,wherein said method further comprising at least one step selected fromthe group consisting of: f culturing the yeast cells in a liquid mediumcomprising wild hawthorn fruit juice and gastric juice of a mammal in asixth electromagnetic field having a frequency in the range of 12,451 to12,460 MHz and a field strength in the range of 280 to 300 mV/cm; and gculturing the yeast cells in a liquid medium comprising wild hawthornfruit juice and gastric juice of a mammal in a seventh electromagneticfield having a frequency in the range of 12,696 to 12,705 MHz and afield strength in the range of 280 to 300 mV/cm.
 3. The method of claim2, further comprising the following steps, which can be carried out inany order: h culturing the yeast cells in a first liquid medium and aneighth electromagnetic field or series of electromagnetic fields havinga frequency in the range of 12,451 to 12,460 MHz and a field strength inthe range of 250 to 340 mV/cm; and i culturing the yeast cells in asecond liquid medium and a ninth electromagnetic field or series ofelectromagnetic fields having a frequency in the range of 12,696 to12,705MHz and a field strength in the range of 250 to 350 mV/cm, whereinthe first and second liquid mediums each comprises wild hawthorn fruitjuice, jujube fruit juice, wu wei zi berry juice, and soybean juice. 4.The method of claim 1, wherein the yeast cells to be treated are cellsof Saccharomyces.
 5. The method of claim 1, wherein the yeast cells tobe treated are cells of Saccharomyces cerevisiae AS2.502.
 6. The methodof claim 2 or 3, wherein the yeast cells to be treated are cells ofSaccharomyces.
 7. The method of claim 2 or 3, wherein the yeast cells tobe treated are cells of Saccharomyces cerevisiae AS2.502.
 8. The methodof claim 2 or 3, further comprising before the recovering step thefollowing steps: j drying the yeast cells at a temperature not exceeding65° C. for a period of time, wherein the yeast cells become dormant; andk drying the yeast cells at a temperature not exceeding 70° C. for aperiod of time to reduce the moisture content to below 5%.